Serine integrase chimeras with activity in E. coli and HeLa cells.

Alfonso P Farruggio,Michele P Calos

doi:10.1242/bio.20148748

Abstract

ABSTRACTIn recent years, application of serine integrases for genomic engineering has increased in popularity. The factor-independence and unidirectionality of these large serine recombinases makes them well suited for reactions such as site-directed vector integration and cassette exchange in a wide variety of organisms. In order to generate information that might be useful for altering the specificity of serine integrases and to improve their efficiency, we tested a hybridization strategy that has been successful with several small serine recombinases. We created chimeras derived from three characterized members of the serine integrase family, phiC31, phiBT1, and TG1 integrases, by joining their amino- and carboxy-terminal portions. We found that several phiBT1-phiC31 (BC) and phiC31-TG1 (CT) hybrid integrases are active in E. coli. BC chimeras function on native att-sites and on att-sites that are hybrids between those of the two donor enzymes, while CT chimeras only act on the latter att-sites. A BC hybrid, BC{−1}, was also active in human HeLa cells. Our work is the first to demonstrate chimeric serine integrase activity. This analysis sheds light on integrase structure and function, and establishes a potentially tractable means to probe the specificity of the thousands of putative large serine recombinases that have been revealed by bioinformatics studies.

Highlights

Serine integrases mediate recombination between two distinct,50 bp phage and bacterial sequences named attP and attB, respectively (Brown et al, 2011; Smith et al, 2010)
We show that they can operate in E. coli on parental and/or chimeric att-sites, and that a select few are able to function in HeLa cells
Three of the four attempted hybrid architectures yielded chimeras with at least marginal activity in E. coli (CT, TC and BC; Fig. 7A). Only two of these hybrid enzyme classes supported full catalytic domain substitutions (CT and BC), and only one chimeric architecture was robustly active in both E.coli and HeLa (BC; Fig. 7A,B)

Summary

Introduction

Serine integrases mediate recombination between two distinct ,50 bp phage and bacterial sequences named attP and attB, respectively (Brown et al, 2011; Smith et al, 2010). Without assistance from other proteins, the reaction proceeds in a unidirectional manner to produce the left and right attachment sites – attL and attR (Smith et al, 2010). Because they are ,200– 350 residues larger than the small serine recombinases (Fig. 1), serine integrases are classified as members of the large serine recombinase sub-family (Smith and Thorpe, 2002). The N-terminal domain is principally involved in catalysis, and imparts some sequence

Methods

Results

Conclusion